There is a clinical need for rapid, multiplexed, and cost-effective detection of soluble protein-based cancer biomarkers. Photonic crystal (PC) surfaces have been recently demonstrated as an effective approach for increasing the output and collection efficiency of fluorescent dyes that are used for protein microarray biomarker detection. Using a quantitative sandwich fluorescent enzyme-linked immunosorbent assay (ELISA) format, the combined effects of PC- enhanced fluorophore excitation and PC-enhanced extraction have been used to reduce the limits of detection of breast cancer biomarkers in plasma, compared to performing the same assay on an ordinary glass surface, resulting in the ability to detect biomarkers in the 0.1 - 10 pg/ml concentration range, as is required for low abundance proteins. In the proposed project, we will develop several innovative approaches for the instrument design that will further reduce the limits of detection for PC-based fluorescent ELISA assays, while integrating the PC into a microfluidic format that will minimize assay volume and allow the assay process to be performed on a droplet of plasma. The PC will be fabricated from low autofluorescence silicon materials, with a design that enables a high quality-factor resonant optical mode to simultaneously provide enhanced excitation and enhanced extraction of fluorescence. The detection instrument will utilize a novel laser scanning approach that optimally couples laser illumination into the PC and matches the resonant coupling condition. The proposed effort seeks to translate photonic crystal enhanced fluorescence (PCEF) technology developed to the proof-of-principle stage under previous NIH funding towards clinical applications. In our previous work, PC device structures, fabrication methods, and detection instruments were developed and first demonstrated for DNA microarrays and protein microarrays. While detection of a panel of breast cancer biomarkers will be evaluated in the proposed project, the detection platform can be applied to multiplexed arrays of biomarker assays for other cancers and diseases. To validate the new sensor and detection instrument, we will detect biomarkers spiked into plasma to establish calibration standards, directly compare against conventional ELISAs, and subsequently quantify biomarker concentrations in blood from breast cancer patients with known Her2 and estrogen receptor statuses. A suite of bioinformatics tools will be modified for automated data analysis and interpretation. The overall goal is to develop a highly sensitive, multiplexed, rapid, and automated assay platform for fluorescent microarray ELISA that can perform biomarker analysis on a droplet of plasma.